Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung

Abstract

A sensitive, fixed-time, spectrophotometric assay for angiotensin-converting enzyme measures the rate of production of hippuric acid from hippuryl-L-histidyl-L-leucine (HHL). The angiotensin-converting enzyme from rabbit lung acetone powder extract, when assayed by this method, is optimally active at pH 8.1 to 8.3 at a chloride ion concentration of 300 mM and an HHL concentration of 5-10 mM; the Km for HHL is 2-6 mM. The enzyme was inhibited by metal-chelating agents, heavy metal salts and certain peptides. The most effective inhibitors were EDTA; CdBr2; angiotensin II; bradykinin; and a pentapeptide, L-pyroglutamyl-L-lysyl-L-tryptophyl-L-alanyl-L-proline, a component of Bothrops jararaca venom. Enzyme inhibited by 0.1 mM EDTA was completely reactivated after removal of EDTA by dialysis but, after prolonged dialysis of the enzyme against 1 mM EDTA, reactivation could only be achieved by addition of metal ions: MnCI2 (40%), ZnCl2 (100%) or Co(NO3)2 (160%). The angiotensin-converting enzyme of rabbit lung is a stable, chloride ion-activated metalloenzyme, similar to both the angiotensin-converting enzyme and kininase II of plasma. © 1971.